2.1. INTERACTION OF STEROID AMINES WITH OLIGONUCLEOTIDES
Steroid diamines have been found to exert a variety of effects in a number of biological systems. For example, these substances have been found to exert antimicrobial and mutagenic activity towards bacteriophages and interfere with the excitability of the neuromuscular junction in animals and man (reviewed in Waring and Henley, 1975, Nucl. Acids Res. 2: 567-586).
The interaction of steroid diamines in particular with DNA sequences has been widely studied. (See for example Hui et al., 1989, Nucl. Acids Res. 17: 4177-4187; Gourevitch and Puigdomenech, 1986, Int. J. Biol. Macromol. 8: 97-104; Patel et al., 1981, Proc. Natl. Acad. Sci. U.S.A. 78: 4063-4067; Dattagupta et al., 1978, Proc. Natl. Acad. Sci. U.S.A. 75: 4286-4290; and Warring and Henley, 1975, Nucl. Acids Res. 2: 567-586; and Mahler et al., 1968, Biochemistry 7: 1568-1582). Steroid diamines have been found to attach firmly to poly-anionic double-stranded DNA by electrostatic interactions (Saucier et al., 1978, Biochemistry 16: 5879). To date, no reports of binding of steroid amines to DNA-RNA complexes, RNA-RNA complexes, or oligonucleotide complexes including a triple helix structure have been found.
The steroid diamines consist of a rigid structure, the steroid. The two amines are affixed to the steroid at appropriate positions to allow for one of the amine groups to interact with one phosphate of the duplex, and the other amine group to interact with the second phosphate chain of the duplex via ionic bonds. The two amines and the rigid structure of the steroid diamines allows the steroid diamines to bind to nucleic acids duplexes in a different manner than the binding of such dicationic ligands as polymethylene diamines, e.g. putrescine, cadaverine. Firstly, steroid diamines show the usual rise of melting temperature (Tm) of DNA at a low steroid diamine/DNA ratio (input ratio) observed for other diamines, but the Tm's decrease at higher input ratios (Waring and Henley, 1975, Nucl. Acids Res. 2: 567-586). Secondly, steroid diamines have been found to have a hyperchromic effect and alter the optical rotatory dispersion and circular dichroism spectra of duplex DNA (Mahler et al., 1968, Biochemistry 7: 1568-1582). Thirdly, steroid diamines have been observed to cause the removal and reversal of the supercoiling in closed circular duplex DNA, attributable to the local unwinding of the DNA helix (Waring and Henley, 1975, Nucl. Acid Res. 2: 567-586). The results from extensive physicochemical studies appear to indicate that steroid amines bind to DNA in a nonintercalative manner and that binding of the steroid diamines induces a kink in the structure of DNA. The steroid diamine may partially insert between slightly unstacked base pairs, but does not intercalate. It was recently shown that the steroid diamine, dipyrandium binds preferentially to the minor groove of AT sequences. (Hui et al., 1989, Nucl. Acids Res. 17: 4177-4187).
The interaction of steroid monoamines bearing a single amino substituent at either the 3 or 17 position with circular DNA has also been studied (Waring and Henley, 1975, Nucl. Acids Res. 2: 567-586). It was found that the two 17.beta.-aminoandrostane compounds tested had a positive effect on supercoiling. However, the effects of these monoamines on the Tm were observed to be considerably smaller than those of their diamine counterparts. The 3.beta.-aminoandrostane compounds tested as well as the 17.beta.-aminoandrostene compound were relatively ineffective. The monoamino steroids can only interact with one phosphate chain of a complex at a time, and so little or no stabilization of the complex is observed.